Poly(ethylene glycol) (PEG) is used to mediate cell-cell fusion in the production of somatic cell hybrids and in the fusion injection of macromolecules into cultured cells from erythrocytes or liposomes. In the previous funding period, we defined in greater molecular detail the mechanism of PEG-mediated fusion and showed that it is remarkably similar in kinetically to the kinetic behaviors defined by electrophysiological and fluorescence studies of influenza virus hemagglutinin-mediated fusion of cell membranes and fusion of secretory vesicles with secretory cell surface membranes. This has allowed us to pursue the mechanism of PEG-mediated fusion as a mimetic for much more complex protein-mediated cell membrane fusion. We have determined the optimal lipid composition for PEG-mediated fusion and found this to be nearly the same as the composition of synaptic vesicle membranes. We showed that fusion peptides from influenza virus and HIV fusion machines do not induce fusion but can enhance PEG-mediated fusion of fusogenic membranes. We found that membrane curvature and PEG-associated osmotic stress both had dramatic effects on the kinetics of PEG-mediate fusion. We also developed new assays for lipid movement between membranes and for "hydrophobic mismatch" associated with fusion intermediates. We have recently developed a simple model of the thermodynamics of a proposed reaction profile for the fusion process that suggests that molecular mismatch within the hydrophobic interior of intermediate structures in the fusion pathway may dominate the kinetics of the process. In the next funding period, we will test whether a simple kinetic scheme based on this model can account for the time courses of lipid and trapped aqueous compartment rearrangements during fusion. Next, we plan to use this model as a framework within which to address a unifying hypothesis for how the "fusion peptide" and trans- membrane domains (TND) of fusion proteins contribute to biomembrane fusion. Finally, we will, in conjunction with the structural cell biology lab of Axel Brunger, develop a model system to define how SNARES and associated neuronal or intracellular fusion proteins influence the mechanism of PEG- mediate vesicle fusion. The results will shed light on the mechanisms of such natural cell fusion processes as endocytosis, exocytotic excretion, protein sorting, and viral budding and infection.